The present invention concerns molecules which bind and neutralize the cytokine interferon-gamma. More specifically, the present invention relates to sheep-derived antibodies and engineered antibody constructs, such as humanized single-chain Fv fragments, chimeric antibodies, diabodies, triabodies, tetravalent antibodies and peptabodies which can be used to treat diseases wherein interferon-gamma activity is pathogenic. Examples of such diseases are: septic shock, cachexia, multiple sclerosis and psoriasis.
Interferon-gamma (IFNxcex3) is a member of the interferon family of immunomodulatory proteins and is produced by activated T helper type-1 cells (Th1 cells) and natural killer cells (NK cells). Apart from its potent antiviral activity, IFNxcex3 is known to be involved in a variety of immune functions (for a review, see Billiau, 1996) and inflammatory responses. Indeed, IFNxcex3 is the primary inducer of the expression of the major histocompatibility complex (MHC) class-II molecules (Steinman et al., 1980) by macrophages and other cell types and stimulates the production of inflammatory mediators such as tumor necrosis factor-alpha (TNFxcex1), interleukin-1 (IL-1) and nitric oxide (NO) (Lorsbach et al., 1993). In this respect, IFNxcex3 is shown to be important in the macrophage-mediated defence to various bacterial pathogens. Furthermore, IFNxcex3 is also shown to be a potent inducer of the expression of adhesion molecules, such as the intercellular adhesion molecule-1 (ICAM-1, Dustin et al., 1988), and of important costimulators such as the B7 molecules on professional antigen presenting cells (Freedman et al., 1991). Moreover, IFNxcex3 induces macrophages to become tumoricidal (Pace et al., 1983) and provokes Ig isotype switching (Snapper and Paul, 1987).
The anti-viral, tumoricidal, inflammatory- and immunomodulatory activity of IFNxcex3 clearly has beneficial effects in a number of clinical conditions. However, there are a number of clinical situations in which IFNxcex3-activity has deleterious effects. These include cancer cachexia (Denz et al., 1993; Iwagaki et al., 1995), septic shock (Doherty et al., 1992), skin disorders such as psoriasis and bullous dermatoses (Van den Oord et al., 1995), allograft rejection (Landolfo et al., 1985; Gorczynski, 1995), chronic inflammations such as ulcerative colitis and Crohn""s disease (WO 94/14467 to Ashkenazi and Ward), and autoimmune diseases such as multiple sclerosis (M S, Panitch et al., 1986), experimental lupus (Ozmen et al., 1995), arthritis (Jacob et al., 1989; Boissier et al., 1995) and autoimmune encephalomyelitis (Waisman et al., 1996).
Cachexia is a phenomenon often seen in cancer patients and is associated with losses of lean body mass, and altered carbohydrate and lipid metabolism. This so called xe2x80x98chronic wasting syndromxe2x80x99 is often the immediate cause of death. In recent years, interest has focused on the role of proinflammatory cytokines in cancer related cachexia. Current data support the concept that cachexia is linked to the presence of certain cytokines among which IFNxcex3 seems to play a central role. Denz et al. (1993) reported that increased neopterin and decreased tryptophan concentrationsxe2x80x94which are closely related to IFNxcex3-activityxe2x80x94are detected in cachectic patients suffering from hematological disorders. Neopterin is synthesized and secreted by monocytes/macrophages upon stimulation by IFNxcex3 from activated T cells. Tryptophan is an indispensable amino acid which can be catabolized by indoleamine 2,3-dioxygenase, an enzyme induced by IFN""s, and which absence initiates mechanisms responsible for cachexia (Brown et al., 1991). The correlation between high neopterin levels, decreased tryptophan levels and weight loss was confirmed by Iwagaki et al. (1995). In experimental models, cancer-induced cachexia can be altered by the administration of IFNxcex3 neutralizing antibodies (Matthys et al., 1991; Langstein et al., 1991) Septic shock is the result of a severe bacterial infection, and remains a common cause of death among critically ill, hospitalized patients despite improvements in supportive care (Bone et al., 1992). Although septic shock may be associated with gram-positive infections, attention has focused on the more common pathogenesis of gram-negative sepsis and the toxic role of endotoxin (=lipopolysaccharide or LPS), a component of the outer membrane of gram-negative and some gram-positive bacteria. Many of the effects of LPS are mediated through the release of cytokines such as TNFxcex1 (Tracey, 1991), IL-1 (Wakabayashi et al., 1991) and IFNxcex3 (Bucklin et al., 1994). Much of the evidence supporting the role of these cytokines as mediators of septic shock comes from lethality studies involving the blockade of individual cytokines, resulting in protection of experimental animals from otherwise lethal doses of endotoxin or gram-negative bacteria. One of the first events in septic shock is the activation of T cells by antigen presenting cells onto which bacterial superantigen is bound (Miethke et al., 1993). Upon activation, for which co-stimulation of CD28 is essential (Saha et al., 1996), these T cells proliferate and produce a surge of proinflammatory cytokines such as IL-2, TNFxcex1 and IFNxcex3 eventuating in the clinical syndrome. Also, it is hypothesized that LPS induces the expression of the xcex11/xcex21 integrin (VLA-1) heterodimer on activated monocytes which then display an increased capacity to adhere to the endothelial basement membrane. Similar effects can be induced by incubation of monocytes with IFNxcex3 (Rubio et al., 1995). VLA-1 might also contribute to further monocyte activation and potentiation of the production of monocyte-derived pro-inflammatory cytokines during sepsis (Rubio et al., 1995). Although very promising results were obtained with antibodies neutralising TNFxcex1 in experimental animal models, clinical trials with anti-TNFxcex1 antibodies revealed only a slight reduction or even no reduction in mortality rate of patients with septic shock (Wherry et al., 1993; Reinhart et al., 1996). A fusion protein containing the extracellular portion of the TNF receptor and the Fc portion of IgG1 also did not affect mortality (Fisher et al., 1996). Pentoxifylline (PTX), a methyl xanthine derivative, is currently being tested for its effect on the outcome of septic shock. PTX is known to lower the serum concentrations of at least TNFxcex1, IL-1 and IFNxcex3 (Bienvenu et al., 1995; Zeni et al., 1996). Initial data reveal that PTX leads to an improvement of the clinical status of septic patients (Mandi et al., 1995). There is evidence that IFNxcex3 is a mediator of lethality during sepsis. Antibodies that either neutralize IFNxcex3 or block the IFNxcex3-receptor are protecting against lethality (Bucklin et al., 1994; Doherty et al., 1992). A synergistic effect between IFNxcex3 and TNFxcex1 has also been suggested (Doherty et al., 1992; Ozmen et al., 1994). Although not in itself lethal, IFNxcex3 has been shown to be essential for the manifestation of TNF-induced lethality in the generalized Shwartzman reaction (Ozmen et al., 1994).
Bullous, inflammatory and neoplastic dermatoses are a heterogenous group of skin disorders during which IFNxcex3 may play a pathogenic role. Bullous dermatoses encompass epidermolysis bullosa acquisita, bullous penihigoid, dermatitis herpetiformes Duhring, linear IgA disease, herpes gestationis, cicatricial pemhigoid, bullous systemic lupus erythematosis, epidermolysis bullosa junctionalis, epidermolysis bullosa dystrophicans, porphyria cutanea tarda and Lyell-Syndrome (Megahed, 1996). Also erythema exsudativum multiform major (Kreutzer et al., 1996), IgG-mediated subepidermal bullous dermatosis (Chan and Cooper, 1994), bullous lichen planus (Willsteed et al., 1991) and paraneoplastic bullous dermatosis (Pantaleeva, 1990) can be classified among the bullous dermatoses. A pathogenic role of IFNxcex3 during bullous dermatoses has been suggested by Van den Oord et al. (1995). The role of IFNxcex3 during inflammatory and neoplastic dermatoses, compared to bullous dermatoses, has been more extensively investigated. Indeed, it has been demonstrated that IFNxcex3 is involved during the pathogenesis of verrucosis (Asadullah et al., 1997), eosinophilic pustular folliculitis (Teraki et al., 1996), cutaneous T cell lymphoma (Wood et al., 1994), granuloma faciale (Smoller and Bortz, 1993), Sweet""s syndrome (Reuss-Borst et al., 1993), atopic eczema (Arenberger et al., 1991), follicular mucinosis (Meisnerr et al., 1991), lichen-planus and psoriasis (Vowels et al., 1994). One of the most extensively studied inflammatory dermatoses is psoriasis. Psoriasis is a hyperproliferative skin disorder affecting approximately 2% of the population. Evidence is accumulating that the disease has a T-cell mediated autoimmune etiology. The role of T-cells in psoriasis has been demonstrated by Gottlieb et al. (1995). The latter authors suggested that, in most of the patients, clinical and histopathological features of psoriasis are primarily linked to skin infiltration by IL-2 receptor-positive leukocytes. Disease improvement can be induced by the administration of a fusion protein composed of human interleukin-2 and fragments of diphteria toxin, which selectively blocks the growth of activated lymphocytes. Other effective anti-psoriatic, T-cell suppressing agents include the immunosuppressive drugs cyclosporin and FK506 (Griffiths, 1986) and anti-CD4 monoclonal antibodies (Morel et al., 1992). More direct evidence for the role of T cells in the induction of the complex tissue alterations seen in psoriasis has been generated by Schxc3x6n et al. (1997) using a model with scid/scid mice in which they transferred naive, minor histocompatibility mismatched CD4+ T-cells, resulting in the development of a skin disorder that resembles psoriasis. The autoimmune character of the disease has been proposed by Valdimarsson et al. (1995) who stated that products of activated T-cells can induce keratinocytes of individuals with psoriatic predisposition to express determinants that are recognized by T cells specific for epitopes on xcex2-haemolytic streptococci. Several data suggest that IFNxcex3 may play a crucial role in the pathogenesis of psoriasis. IFNxcex3, produced by activated T cells would be involved in the recruitment of lymphocytes (Nickoloff, 1988), in the induction of activation and adhesion molecules on epidermal keratinocytes (Dustin et al., 1988), as well as in the abnormal keratinocyte proliferation (Barker et al., 1993). Not only enhanced levels of IFNxcex3 has been detected in psoriatic epidermis (Kaneko et al., 1990), also de novo suprabasal expression of IFNxcex3 receptor in psoriasis has been demonstrated (Van den Oord et al., 1995).
Inflammatory bowel disease (IBD), which encompasses ulcerative colitis and Crohn""s disease, is characterized by the appearance of lesions of unknown aetiology in most parts of the gut. IBD is rather common, with a prevalence in the range of 70-170 in a population of 100,000. The current therapy of IBD involves the administration of anti-inflammatory or immunosuppressive agents, which usually bring only partial results, and surgery. In view of the apparent shortcomings of the present treatment, Ashkenazi and Ward (WO 94/14467) suggested the usage of a bispecific antibody construct targeting IFNxcex3 and another molecule, such as IL-1 and TNFxcex1, to treat IBD. However, the exact role of IFNxcex3 during IBD is not well understood.
MS is a severely disabling progressive neurological disease of unknown aetiology, but probably involving autoimmune responses and resulting in the appearance of focal areas of demyelinisation (Williams et al., 1994). MS affects 1 in 1000 persons in the USA and Europe, but due to improved diagnosis that number is increasing. Onset of disease is usually around 30 years of age and, on average, patients are in need of treatment for another 28 years. MS is among the most expensive chronic diseases of western society, based on duration and intensity of care. However, diagnosis of exacerbations and early identification of onset of exacerbations has improved greatly, allowing design of novel treatment strategies. Active multiple sclerosis lesions feature T-lymphocyte and monocyte-macrophage accumulations at plaque margins where myelin is being destroyed. The inflammatory cells that invade the white matter and the soluble mediators that they release are held primarily responsible for myelin breakdown. Population-based studies indicate that certain HLA-antigens occur with higher frequency in patients with MS (with predominant MHC being the Dw2(DR2)DQ1.2 haplotype (Olerup et al., 1991). Similar associations of class I and class II haplotypes have also been detected in other autoimmune disorders such as rheumatoid arthritis and insulin dependent diabetes (Nepom, 1993). The lesions of MS are comparable to those found in chronic relapsing experimental allergic encephalitis (EAE), an autoimmune disease that can be induced in animals by immunization with e.g. whole myelin (Allen et al., 1993) or with the myelin/oligodendrocyte glycoprotein (Genain et al., 1995b). The lesions associated with EAE are similar in appearance as the ones occurring in MS and also contain inflammatory infiltrates of T-cells and macrophages (Genain et al., 1995b). Furthermore, in adoptive transfer experiments, T cells sensitized to specific myelin antigens can transfer the disease state of EAE (Genain et al., 1995b; Waldburger et al., 1996). A few years ago, the American FDA approved the use of the immunosuppressive drug interferon (trade name Betaseron) for treatment of chronic relapsing MS. The effect of this drugxe2x80x94although modestxe2x80x94clearly demonstrates the involvement of the cytokine network in the pathophysiology of MS. In the last few years, a large number of studies have addressed the molecular mechanism by which Betaseron exerts its beneficial effects. Lately, it was shown that IFNxcex2 dose-dependently inhibited T-cell proliferation, expression of IL-2 receptors and secretion of IFNxcex3, TNFxcex1 and IL-13 (Rep et al., 1996). Furthermore, it was demonstrated that IFNxcex2 could specifically prevent the IFNxcex3-induced up regulation of MHC class II antigens and adhesion molecules on antigen-presenting cells (Jiang et al., 1995) and human brain microvessel endothelial cells (Huynh et al., 1995).
One of the earliest events in MS is damage of the blood brain barrier (BBB) by activated, encephalitogenic T-cells (Tsukada et al., 1993). The mechanism by which these cells destruct locally the BBB, which is mainly constituted of endothelial cells, is not elucidated, but it is known that at the systemic level, local production of certain cytokines such as IFNxcex3 enhance the capability of lymphocytes to adhere to endothelial cells (Yu et al., 1985; Tsukada et al., 1993). Also, on choroid plexus epithelial cells of EAE animals, an increased expression of ICAM-1 and VCAM-1 (Steffen et al., 1994), for which LFA-1 and VLA-4 are the natural ligands on lymphocytes, has been observed. Mc Carron et al. (1993) reported that adhesion of MBP-specific T lymphocytes was significantly up regulated when cerebral endothelial cells were treated with IL-1, TNFxcex1 or IFNxcex3. That the adhesion of encephalitogenic T-cells to the endothelium is an early and very important event in the onset of MS is shown by the finding that anti LFA-1 therapy can completely block the induction of EAE (Gordon et al., 1995). Additional circumstantial evidence for a stimulatory role of IFNxcex3 in the pathophysiology of MS comes from observations that disease exacerbations are induced by viral upper respiratory infections, known to stimulate the secretion of IFNxcex3 by type-2 helper T cells (Panitch, 1994). The proinflammatory role of IFNxcex3 in autoimmune disease is strengthened by an earlier finding that treatment of MS patients with hIFNxcex3 resulted in an aggravation of the symptoms (Panitch et al., 1986). The role of IFNxcex3 as proinflammatory cytokine in autoimmune disorders has been studied in several experimentally induced forms of autoimmunity. In experimental neuritis, induced by myelin or antigen-specific T cells in rat, IFNxcex3 clearly acted as pro-inflammatory cytokine and administration of a monoclonal antibody to IFNxcex3 suppressed the disease (Hartung et al., 1990). In the case of experimental autoimmune thyroiditis (EAT) in mice, induced by the injection of thyroglobulin, treatment of the animals with anti-IFNxcex3 at 4 weeks after induction of EAT proved to be beneficial, since characteristic features of EAT such as the lymphocytic infiltrations of the thyroid glands and the serum levels of autoantibodies to thyroglobulin, were significantly reduced (Tang et al., 1993).
In the mouse EAE model for MS, where the disease can be induced by injection of either spinal cord homogenate or myelin basic protein, elevated concentrations of several cytokines, including IFNxcex3 were observed both in serum and in the lesions in the CNS (Willenborg et al., 1995). However, administration of anti-IFNxcex3 at the initiation of the disease, resulted in an exacerbation of the disease (Billiau et al., 1988; Duong et al., 1994; Willenborg et al., 1995). It must be noted, however, that in these experiments the effect of anti-IFNxcex3 was determined at the onset of acute EAE rather than at the time of chronic relapse of the disease, which in fact is the only relevant situation for MS. Pathologically, typical acute EAE differs substantially from MS in that prominent inflammation occurs in gray, white and meningeal structures, but demyelisation is scant or absent (Genain et al., 1995b). In order to explain the findings with anti-IFNxcex3antibodies, the authors suggest a different action of IFNxcex3 at the systemic level (anti-inflammatory action) compared to the local level (inflammatory action) (Billiau et al., 1988), or suggest an early role (within 24 h after immunization) of IFNxcex3 in disease resistance (Duong et al., 1994). Willenborg et al. (1995) conclude that the time of treatment plays a critical role on the outcome and suggest this to be the explanation for conflicting results in different autoimmune processes. Recently, Heremans et al. (1996) described facilitation of spontaneous relapses in chronic relapsing EAE in Biozzi ABH mice by administration of anti-IFNxcex3 during the remission phase. The onset of relapses was delayed when animals were treated with IFNxcex3 during the remission phase, results which are in contradiction to the excacerbation seen in humans who were treated with hIFNxcex3.
An experimental EAE model that more closely resembles the disease course and symptomatology of MS in humans can be found in marmosets. Indeed, in these animals a chronic relapsing-remitting form of EAE can be induced which is characterized by an initial, acute phase with clinically mild neurological signs, followed by recovery. A late spontaneous relapse occurs in these animals and chronic lesions resemble active plaques of chronic MS (Massacesi et al., 1995). This unique model can efficiently be employed to evaluate a prospective therapy for MS. In this model, a critical role for TNFxcex1 in demyelisation is suggested by the observation that rolipram, a selective inhibitor of the type IV phosphodiesterase, suppressed TNFxcex1 secretion and demyelisation (Genain et al., 1995a; Sommer et al., 1995) when administered shortly after immunization, thus interfering with acute EAE. The effect of anti-IFNxcex3 on acute EAE or on disease relapse has to our knowledge never been investigated in marmoset.
Taken together, it is well established that there are a number of clinical situations in which IFNxcex3-activity has deleterious effects. Consequently, several potential therapies to neutralize IFNxcex3-activity have been proposed. Among the latter proposals are the use of: anti-IFNxcex3 antibodies (Ozmen et al., 1995; Bucklin et al., 1994), recombinant anti-IFNxcex3 Fv fragments (EP 0528469 to Billiau and Froyen), bispecific molecules (WO 94/14467 to Ashkenazi and Ward), drugs such as pentoxifylline (Bienvenu et al., 1995), synthetic polypeptides which inhibit binding of IFNxcex3 to its receptor (U.S. Pat No. 5,451,658 to Seelig; U.S. Pat. No. 5,632,988 to Ingram et al.), Epstein-Barr virus derived proteins (U.S. Pat. No. 5,627,155 to Moore and Kastelein), soluble IFNxcex3 receptors (EP 0393502 to Fountoulakis et al.; U.S. Pat. No. 5,578,707 to Novick and Rubinstein) and oligonucleotides which bind to IFNxcex3(WO95/00529 to Coppola et al.). However, these compounds are faced with problems such as suboptimal stability, affinity and clearance rates, lack of specificity, efficacy and tissue penetrance, toxic side effects and unwanted carrier effects. Indeed, the carrier effect of antibodies can limit their efficiency to block the target cytokine. For example, Montero-Julian et al. (1995) showed that during treatment of myeloma patients with anti-IL-6, accumulation of IL-6 in the serum in the form of monomeric immune complexes occurred, hereby stabilizing the cytokine. Furthermore, it has also been shown that the therapeutic efficacy of a cytokine can be prolonged by the formation of cytokine/antibody complexes, since the efficacy of recombinant human IL-2 treatment could be increased by prolonging its in vivo half-life by complexing with an anti-IL-2 antibody (Courtney et al., 1994). The carrier-effect of anti-cytokine antibodies can be overcome by the construction of monovalent scFv fragments, although their low MW (V30.000) and the associated fast clearance rate, make them less suitable candidates for long-term treatment. However, the undesirable carrier effect can be avoided by the formation of higher immune complexes, as such increasing the clearance of the cytokine-antibody complexes (Montero-Julian et al., 1995). The use of monoclonal antibodies for diagnostic or therapeutic purposes in vivo is, besides the carrier effect, also limited because of their nature (i.e. the majority are murine mAb""s and administration of antibodies of mouse origin inevitably results in a human anti-mouse antibody [HAMA] response), their suboptimal efficacy, stability and affinity and their large molecular size. Proposed solutions to some of these problems involve the use of F(abxe2x80x2)2, F(ab) and scFv derivatives or of humanized versions of the parent antibody, either by CDR grafting (Kettleborough et al., 1991) or by resurfacing of the antibodies (Roguska et al., 1994). Another proposed solution is the development of several modified antibodies or antibody constructs by bioengineering or chemical methods. Indeed, some mAb""s were made more effective by conjugating chemotherapeutic drugs and other toxins to the antibodies (Ghetie and Vitetta, 1994) or by developing bispecific and/or multivalent antibody constructs capable of simultaneously binding severalxe2x80x94or two different epitopes on the samexe2x80x94or different antigens. These antibody constructs have been produced using a variety of methods: a) antibodies of different specificities or univalent fragments of pepsin-treated antibodies of different specificities have been chemically linked (Fanger et al., 1992); b) two hybridomas secreting antibodies of different specificity have been fused and the resulting bispecific antibodies from the mixture of antibodies were subsequently isolated; c) genitically engineered single chain antibodies have been used to produce non-covalently linked bispecific antibodies (e.g. diabodies (Holliger et al., 1993), minibodies (Kostelny et al., 1992) and tetravalent antibodies (Pack et al; 1995; WO 96/13583 to Pack) or covalently-linked bispecific antibodies (e.g. chelating recombinant antibodies (Kranz et al., 1995), single chain antibodies fused to protein A or Streptavidin (Ito and Kurosawa, 1993; Kipriyanov et al., 1996) and bispecific tetravalent antibodies (EP 0517024 to Bosslet and Deeman). Recently, also trivalent antibody constructs, named triabodies (Kortt et al., 1997), and pentavalent constructs, named peptabodies (Terskikh et al., 1997), have been described. These constructs may have a higher avidity in comparison to bivalent constructs and may be useful for diagnostic or therapeutic purposes in vivo.
However, and despite the fact that several potential therapies to neutralize IFNxcex3-activity have been proposed, no prior art exists regarding the production and existence of engineered antibody constructs, such as humanized single-chain Fv fragments, diabodies, triabodies, tetravalent antibodies, peptabodies and hexabodies, and ruminant-derived antibodies such as sheep antibodies which overcome the above-indicated problems and which can efficiently be used to treat diseases wherein interferon-gamma activity is pathogenic.
It is clear from the prior art as cited above that problems such as suboptimal stability, affinity, clearance rate, specificity, efficacy, and an unwanted carrier effect and HAMA response hamper the successful usage of several therapeutics which, potentially, could neutralize the activity of IFNxcex3. Also suggested solutions to overcome some of these problems did not result in the development of effective products. Thus, unpredictable and unknown factors still appear to determine the success of these biologicals. Despite these unknown factors, the present inventors have been able to design and develop useful constructs which effectively neutralize IFNxcex3-activity. Indeed, the constructs have all a surprisingly high affinity for IFNxcex3, they do not provoke a HAMA or related response, and they do not result in a carrier effect. In addition, some of the constructs pass the blood brain barrier, whereas others have a very good clearance rate. Therefore, the present invention aims at providing a molecule which binds and neutralizes interferon-gamma and which is chosen from the group consisting of:
a scFv comprising the humanized variable domain of the monoclonal antibody D9D10
a chimeric antibody comprising the humanized variable domain of the monoclonal antibody D9D10
a diabody comprising the humanized variable domain of the monoclonal antibody D9D10
a multivalent antibody
a ruminant antibody.
The present invention further aims at providing a multivalent antibody chosen from the group consisting of triabodies, tetravalent antibodies, peptabodies and hexabodies.
The present invention also aims at providing a triabody, tetravalent antibody, peptabody and hexabody which comprise 3, 4, 5 and 6 variable domains, respectively, of different anti-interferon-gamma antibodies.
The present invention further aims at providing a triabody as described above which comprises 3 identical variable domains of an anti-interferon-gamma antibody. A preferred variable domain used in the latter constructs is derived from the mouse anti-interferon-gamma antibody D9D10 which is described by Sandvig et al. (1987) and Froyen et al. (1993) or from the sheep anti-interferon-gamma antibody described in the present application. Therefore, the present invention aims at providing a triabody as described above which comprises 3 identical D9D10 scFv""s, 3 identical humanized D9D10 scFv""s, 3 identical sheep-derived anti-interferon-gamma scFv""s or 3 identical humanized sheep-derived anti-interferon-gamma scFv""s.
The present invention further aims at providing a tetravalent antibody (called MoTAb I) as described above which comprises 4 identical domains of an anti-interferon-gamma antibody. More specifically, the present invention aims at providing a tetravalent antibody as described above which comprises either 4 identical D9D10 scFv""s or 4 identical sheep-derived anti-interferon-gamma scFv""s in the format of a homodimer of 2 identical molecules, each containing 2 D9D10 scFv""s or 2 humanized D9D10 scFv""s or 2 sheep-derived anti-interferon-gamma scFv""s or 2 humanized sheep-derived anti-interferon-gamma scFv""s, and a dimerization domain, or, a full-size humanized D9D10 antibody or sheep-derived anti-interferon-gamma antibody to which 2 humanized D9D10 scFv""s or 2 humanized sheep-derived anti-interferon-gamma scFv""s, respectively, are attached at the carboxyterminus (called MoTAb II) (see FIG. 1).
The present invention further aims at providing a peptabody and hexabody as described above which comprise 5 and 6 identical variable domains of an anti-interferon-gamma antibody, respectively. A preferred variable domain used in the latter constructs is derived from the mouse anti-interferon-gamma antibody D9D10 which is described above or from the sheep anti-interferon-gamma antibody described in the present application. Therefore, the present invention aims at providing a peptabody and hexabody as described above which comprises 5 or 6 identical D9D10 scFv""s, 5 or 6 identical humanized D9D10 scFv""s, 5 or 6 identical sheep-derived anti-interferon-gamma scFv""s, or, 5 or 6 identical humanized sheep-derived anti-interferon-gamma scFv""s, respectively.
The present invention further aims at providing a molecule as described above, wherein said ruminant antibody is a sheep antibody.
The present invention also aims at providing a molecule as described above, wherein said sheep antibody is a monoclonal antibody. Furthermore, the present invention aims at providing a humanized antibody, a single-chain fragment or any other fragment which is derived from said monoclonal antibody and which has largely retained the specificity of said monoclonal antibody.
Moreover, the present invention aims at providing methods for producing the above-described molecules.
The present invention further aims at providing a pharmaceutical composition comprising a molecule as described above, or a mixture of said molecules, in a pharmaceutically acceptable excipient.
The present invention also aims at providing a molecule or a composition as described above for use as a medicament.
Furthermore, the present invention aims at providing a molecule or a composition as described above for preventing or treating septic shock, cachexia, immune diseases such as multiple sclerosis and Crohn""s disease and skin disorders such as bullous, inflammatory and neoplastic dermatosis.
Finally, the present invention aims at providing a molecule as described above for determining interferon gamma levels in a sample.
All the aims of the present invention are considered to have been met by the embodiments as set out below.